RNA-guided human genome engineering

ABSTRACT

A method of altering a eukaryotic cell is provided including transfecting the eukaryotic cell with a nucleic acid encoding RNA complementary to genomic DNA of the eukaryotic cell, transfecting the eukaryotic cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site specific manner, wherein the cell expresses the RNA and the enzyme, the RNA binds to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site specific manner.

RELATED APPLICATION DATA

This application is a continuation application which claims priority toU.S. patent application Ser. No. 14/653,144, filed on Jun. 17, 2015,which is a National Stage Application under 35 U.S.C. 371 of co-pendingPCT application PCT/US2013/075317, filed Dec. 16, 2013 and U.S.provisional application No. 61/799,169, filed Mar. 13, 2013 and U.S.provisional application No. 61/738,355, filed Dec. 17, 2012 each ofwhich is hereby incorporated herein by reference in their entirety forall purposes.

STATEMENT OF GOVERNMENT INTERESTS

This invention was made with government support under P50 HG005550awarded by National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

Bacterial and archaeal CRISPR systems rely on crRNAs in complex with Casproteins to direct degradation of complementary sequences present withininvading viral and plasmid DNA (1-3). A recent in vitro reconstitutionof the S. pyogenes type II CRISPR system demonstrated that crRNA fusedto a normally trans-encoded tracrRNA is sufficient to direct Cas9protein to sequence-specifically cleave target DNA sequences matchingthe crRNA (4).

SUMMARY

The present disclosure references documents numerically which are listedat the end of the present disclosure. The document corresponding to thenumber is incorporated by reference into the specification as asupporting reference corresponding to the number as if fully cited.

According to one aspect of the present disclosure, a eukaryotic cell istransfected with a two component system including RNA complementary togenomic DNA and an enzyme that interacts with the RNA. The RNA and theenzyme are expressed by the cell. The RNA of the RNA/enzyme complex thenbinds to complementary genomic DNA. The enzyme then performs a function,such as cleavage of the genomic DNA. The RNA includes between about 10nucleotides to about 250 nucleotides. The RNA includes between about 20nucleotides to about 100 nucleotides. According to certain aspects, theenzyme may perform any desired function in a site specific manner forwhich the enzyme has been engineered. According to one aspect, theeukaryotic cell is a yeast cell, plant cell or mammalian cell. Accordingto one aspect, the enzyme cleaves genomic sequences targeted by RNAsequences (see references (4-6)), thereby creating a genomically alteredeukaryotic cell.

According to one aspect, the present disclosure provides a method ofgenetically altering a human cell by including a nucleic acid encodingan RNA complementary to genomic DNA into the genome of the cell and anucleic acid encoding an enzyme that performs a desired function ongenomic DNA into the genome of the cell. According to one aspect, theRNA and the enzyme are expressed. According to one aspect, the RNAhybridizes with complementary genomic DNA. According to one aspect, theenzyme is activated to perform a desired function, such as cleavage, ina site specific manner when the RNA is hybridized to the complementarygenomic DNA. According to one aspect, the RNA and the enzyme arecomponents of a bacterial Type II CRISPR system.

According to one aspect, a method of altering a eukaryotic cell isproviding including transfecting the eukaryotic cell with a nucleic acidencoding RNA complementary to genomic DNA of the eukaryotic cell,transfecting the eukaryotic cell with a nucleic acid encoding an enzymethat interacts with the RNA and cleaves the genomic DNA in a sitespecific manner, wherein the cell expresses the RNA and the enzyme, theRNA binds to complementary genomic DNA and the enzyme cleaves thegenomic DNA in a site specific manner According to one aspect, theenzyme is Cas9 or modified Cas9 or a homolog of Cas9. According to oneaspect, the eukaryotic cell is a yeast cell, a plant cell or a mammaliancell. According to one aspect, the RNA includes between about 10 toabout 250 nucleotides. According to one aspect, the RNA includes betweenabout 20 to about 100 nucleotides.

According to one aspect, a method of altering a human cell is providedincluding transfecting the human cell with a nucleic acid encoding RNAcomplementary to genomic DNA of the eukaryotic cell, transfecting thehuman cell with a nucleic acid encoding an enzyme that interacts withthe RNA and cleaves the genomic DNA in a site specific manner, whereinthe human cell expresses the RNA and the enzyme, the RNA binds tocomplementary genomic DNA and the enzyme cleaves the genomic DNA in asite specific manner. According to one aspect, the enzyme is Cas9 ormodified Cas9 or a homolog of Cas9. According to one aspect, the RNAincludes between about 10 to about 250 nucleotides. According to oneaspect, the RNA includes between about 20 to about 100 nucleotides.

According to one aspect, a method of altering a eukaryotic cell at aplurality of genomic DNA sites is provided including transfecting theeukaryotic cell with a plurality of nucleic acids encoding RNAscomplementary to different sites on genomic DNA of the eukaryotic cell,transfecting the eukaryotic cell with a nucleic acid encoding an enzymethat interacts with the RNA and cleaves the genomic DNA in a sitespecific manner, wherein the cell expresses the RNAs and the enzyme, theRNAs bind to complementary genomic DNA and the enzyme cleaves thegenomic DNA in a site specific manner According to one aspect, theenzyme is Cas9. According to one aspect, the eukaryotic cell is a yeastcell, a plant cell or a mammalian cell. According to one aspect, the RNAincludes between about 10 to about 250 nucleotides. According to oneaspect, the RNA includes between about 20 to about 100 nucleotides.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee. The foregoing and other features and advantages ofthe present embodiments will be more fully understood from the followingdetailed description of illustrative embodiments taken in conjunctionwith the accompanying drawings in which:

FIG. 1A depicts genome editing in human cells using an engineered typeII CRISPR system (SEQ ID NO:17 and 45-46). FIG. 1B depicts a genomicallyintegrated GFP coding sequence disrupted by the insertion of a stopcodon and a 68 bp genomic fragment from the AAVS1 locus (SEQ ID NO:18).FIG. 1C-1 bar graph depicts HR efficiencies induced by T1, T2, andTALEN-mediated nuclease activity at the target locus, as measured byFACS. FIG. 1C-2 depicts FACS plots and microscopy images of the targetedcells.

FIG. 2A depicts RNA-guided genome editing of the native AAVS1 locus inmultiple cell types (SEQ ID NO:19). FIG. 2B-1, FIG. 2B-2, and FIG. 2B-3depict total count and location of deletions caused by NHEJ in 293 Ts,K562s, and PGP1 iPS cells. FIG. 2C depicts DNA donor architecture for HRat the AAVS1 locus, and the locations of sequencing primers (arrows) fordetecting successful targeted events. FIG. 2D depicts PCR assay threedays post transfection. FIG. 2E depicts successful HR confirmed bySanger sequencing of the PCR amplicon (SEQ ID NOs:20-21). FIG. 2Fdepicts images of targeted clones of 293T cells.

FIG. 3A-1 and FIG. 3A-2 depict a human codon-optimized version of theCas9 protein and full sequence of the cas9 gene insert (SEQ ID NO:22).FIG. 3B depicts U6 promoter based expression scheme for the guide RNAsand predicted RNA transcript secondary structure (SEQ ID NO:23). FIG. 3Cdepicts targeted GN₂₀GG genomic sites and selected gRNAs (SEQ IDNOs:24-31).

FIG. 4 depicts that all possible combinations of the repair DNA donor,Cas9 protein, and gRNA were tested for their ability to effectsuccessful HR in 293 Ts.

FIG. 5A-5B depict the analysis of gRNA and Cas9 mediated genome editing(SEQ ID NO:19).

FIGS. 6A-6B depict 293T stable lines each bearing a distinct GFPreporter construct (SEQ ID NOs:32-34).

FIG. 7 depicts gRNAs targeting the flanking GFP sequences of thereporter described in FIG. 1B (in 293 Ts).

FIGS. 8A-8B depict 293T stable lines each bearing a distinct GFPreporter construct (SEQ ID NOs:35-36).

FIGS. 9A-9C depict human iPS cells (PGP1) that were nucleofected withconstructs (SEQ ID NO:19).

FIGS. 10A-10B depict RNA-guided NHEJ in K562 cells (SEQ ID NO:19).

FIG. 11A-11B depict RNA-guided NHEJ in 293T cells (SEQ ID NO:19).

FIGS. 12A-12C depict HR at the endogenous AAVS1 locus using either adsDNA donor or a short oligonucleotide donor (SEQ ID NOs:37-38).

FIG. 13A-1 and FIG. 13A-2 and FIG. 13B depict the methodology formultiplex synthesis, retrieval and U6 expression vector cloning of guideRNAs targeting genes in the human genome (SEQ ID NOs:39-41).

FIGS. 14A-14D depict CRISPR mediated RNA-guided transcriptionalactivation (SEQ ID NOs:42-43).

FIGS. 15A-15B depict gRNA sequence flexibility and applications thereof(SEQ ID NO:44-46).

DETAILED DESCRIPTION

According to one aspect, a human codon-optimized version of the Cas9protein bearing a C-terminus SV40 nuclear localization signal issynthesized and cloned into a mammalian expression system (FIG. 1A andFIGS. 3A-1 and 3A-2). Accordingly, FIG. 1 is directed to genome editingin human cells using an engineered type II CRISPR system. As shown inFIG. 1A, RNA-guided gene targeting in human cells involves co-expressionof the Cas9 protein bearing a C-terminus SV40 nuclear localizationsignal with one or more guide RNAs (gRNAs) expressed from the human U6polymerase III promoter. Cas9 unwinds the DNA duplex and cleaves bothstrands upon recognition of a target sequence by the gRNA, but only ifthe correct protospacer-adjacent motif (PAM) is present at the 3′ end.Any genomic sequence of the form GN₂₀GG can in principle be targeted. Asshown in FIG. 1B, a genomically integrated GFP coding sequence isdisrupted by the insertion of a stop codon and a 68 bp genomic fragmentfrom the AAVS1 locus. Restoration of the GFP sequence by homologousrecombination (HR) with an appropriate donor sequence results in GFPcells that can be quantitated by FACS. T1 and T2 gRNAs target sequenceswithin the AAVS1 fragment. Binding sites for the two halves of the TALeffector nuclease heterodimer (TALEN) are underlined. As shown in FIG.1C-1, bar graph depict HR efficiencies induced by T1, T2, andTALEN-mediated nuclease activity at the target locus, as measured byFACS. Representative FACS plots and microscopy images of the targetedcells, FIG. 1C-2, are depicted below (scale bar is 100 microns). Data ismean+/−SEM (N=3).

According to one aspect, to direct Cas9 to cleave sequences of interest,crRNA-tracrRNA fusion transcripts are expressed, hereafter referred toas guide RNAs (gRNAs), from the human U6 polymerase III promoter.According to one aspect, gRNAs are directly transcribed by the cell.This aspect advantageously avoids reconstituting the RNA processingmachinery employed by bacterial CRISPR systems (FIG. 1A and FIG. 3B)(see references (4, 7-9)). According to one aspect, a method is providedfor altering genomic DNA using a U6 transcription initiating with G anda PAM (protospacer-adjacent motif) sequence -NGG following the 20 bpcrRNA target. According to this aspect, the target genomic site is inthe form of GN₂₀GG (See FIG. 3C).

According to one aspect, a GFP reporter assay (FIG. 1B) in 293T cellswas developed similar to one previously described (see reference (10))to test the functionality of the genome engineering methods describedherein. According to one aspect, a stable cell line was establishedbearing a genomically integrated GFP coding sequence disrupted by theinsertion of a stop codon and a 68 bp genomic fragment from the AAVS1locus that renders the expressed protein fragment non-fluorescent.Homologous recombination (HR) using an appropriate repair donor canrestore the normal GFP sequence, which allows one to quantify theresulting GFP cells by flow activated cell sorting (FACS).

According to one aspect, a method is provided of homologousrecombination (HR). Two gRNAs are constructed, T1 and T2, that targetthe intervening AAVS1 fragment (FIG. 1b ). Their activity to that of apreviously described TAL effector nuclease heterodimer (TALEN) targetingthe same region (see reference (11)) was compared. Successful HR eventswere observed using all three targeting reagents, with gene correctionrates using the T1 and T2 gRNAs approaching 3% and 8% respectively (FIG.1C-1). This RNA-mediated editing process was notably rapid, with thefirst detectable GFP cells appearing ˜20 hours post transfectioncompared to ˜40 hours for the AAVS1 TALENs. HR was observed only uponsimultaneous introduction of the repair donor, Cas9 protein, and gRNA,confirming that all components are required for genome editing (FIG. 4).While no apparent toxicity associated with Cas9/crRNA expression wasnoted, work with ZFNs and TALENs has shown that nicking only one strandfurther reduces toxicity. Accordingly, a Cas9D10A mutant was tested thatis known to function as a nickase in vitro, which yielded similar HR butlower non-homologous end joining (NHEJ) rates (FIG. 5) (see references(4, 5)). Consistent with (4) where a related Cas9 protein is shown tocut both strands 6 bp upstream of the PAM, NHEJ data confirmed that mostdeletions or insertions occurred at the 3′ end of the target sequence(FIG. 5B). Also confirmed was that mutating the target genomic siteprevents the gRNA from effecting HR at that locus, demonstrating thatCRISPR-mediated genome editing is sequence specific (FIGS. 6A and 6B).It was showed that two gRNAs targeting sites in the GFP gene, and alsothree additional gRNAs targeting fragments from homologous regions ofthe DNA methyl transferase 3a (DNMT3a) and DNMT3b genes could sequencespecifically induce significant HR in the engineered reporter cell lines(FIG. 7, 8). Together these results confirm that RNA-guided genometargeting in human cells induces robust HR across multiple target sites.

According to certain aspects, a native locus was modified. gRNAs wereused to target the AAVS1 locus located in the PPP1R12C gene onchromosome 19, which is ubiquitously expressed across most tissues (FIG.2A) in 293 Ts, K562s, and PGP1 human iPS cells (see reference (12)) andanalyzed the results by next-generation sequencing of the targetedlocus. Accordingly, FIG. 2 is directed to RNA-guided genome editing ofthe native AAVS1 locus in multiple cell types. As shown in FIG. 2A, T1(red) and T2 (green) gRNAs target sequences in an intron of the PPP1R12Cgene within the chromosome 19 AAVS1 locus. As shown in FIGS. 2B-1, 2B-2,and 2B-3, total count and location of deletions caused by NHEJ in 293Ts, K562s, and PGP1 iPS cells following expression of Cas9 and either T1or T2 gRNAs as quantified by next-generation sequencing is provided. Redand green dash lines demarcate the boundaries of the T1 and T2 gRNAtargeting sites. NHEJ frequencies for T1 and T2 gRNAs were 10% and 25%in 293T, 13% and 38% in K562, and 2% and 4% in PGP1 iPS cells,respectively. As shown in FIG. 2C, DNA donor architecture for HR at theAAVS1 locus, and the locations of sequencing primers (arrows) fordetecting successful targeted events, are depicted. As shown in FIG. 2D,PCR assay three days post transfection demonstrates that only cellsexpressing the donor, Cas9 and T2 gRNA exhibit successful HR events. Asshown in FIG. 2E, successful HR was confirmed by Sanger sequencing ofthe PCR amplicon showing that the expected DNA bases at both thegenome-donor and donor-insert boundaries are present. As shown in FIG.2F, successfully targeted clones of 293T cells were selected withpuromycin for 2 weeks. Microscope images of two representative GFP+clones is shown (scale bar is 100 microns).

Consistent with results for the GFP reporter assay, high numbers of NHEJevents were observed at the endogenous locus for all three cell types.The two gRNAs T1 and T2 achieved NHEJ rates of 10 and 25% in 293 Ts, 13and 38% in K562s, and 2 and 4% in PGP1-iPS cells, respectively (FIGS.2B-1, 2B-2, and 2B-3). No overt toxicity was observed from the Cas9 andcrRNA expression required to induce NHEJ in any of these cell types(FIGS. 9A, 9B, and 9C). As expected, NHEJ-mediated deletions for T1 andT2 were centered around the target site positions, further validatingthe sequence specificity of this targeting process (FIGS. 9A-C, 10, 11).Simultaneous introduction of both T1 and T2 gRNAs resulted in highefficiency deletion of the intervening 19 bp fragment (FIGS. 10A and10B), demonstrating that multiplexed editing of genomic loci is feasibleusing this approach.

According to one aspect, HR is used to integrate either a dsDNA donorconstruct (see reference (13)) or an oligo donor into the native AAVS1locus (FIG. 2C, FIG. 12C). HR-mediated integration was confirmed usingboth approaches by PCR (FIG. 2D, FIG. 12A) and Sanger sequencing (FIG.2E). 293T or iPS clones were readily derived from the pool of modifiedcells using puromycin selection over two weeks (FIG. 2F, FIG. 12B).These results demonstrate that Cas9 is capable of efficientlyintegrating foreign DNA at endogenous loci in human cells. Accordingly,one aspect of the present disclosure includes a method of integratingforeign DNA into the genome of a cell using homologous recombination andCas9.

According to one aspect, an RNA-guided genome editing system is providedwhich can readily be adapted to modify other genomic sites by simplymodifying the sequence of the gRNA expression vector to match acompatible sequence in the locus of interest. According to this aspect,190,000 specifically gRNA-targetable sequences targeting about 40.5%exons of genes in the human genome were generated. These targetsequences were incorporated into a 200 bp format compatible withmultiplex synthesis on DNA arrays (see reference (14)) (FIGS. 13A-1 and13A-2). According to this aspect, a ready genome-wide reference ofpotential target sites in the human genome and a methodology formultiplex gRNA synthesis is provided.

According to one aspect, methods are provided for multiplexing genomicalterations in a cell by using one or more or a plurality of RNA/enzymesystems described herein to alter the genome of a cell at a plurality oflocations. According to one aspect, target sites perfectly match the PAMsequence NGG and the 8-12 base “seed sequence” at the 3′ end of thegRNA. According to certain aspects, perfect match is not required of theremaining 8-12 bases. According to certain aspects, Cas9 will functionwith single mismatches at the 5′ end. According to certain aspects, thetarget locus's underlying chromatin structure and epigenetic state mayaffect efficiency of Cas9 function. According to certain aspects, Cas9homologs having higher specificity are included as useful enzymes. Oneof skill in the art will be able to identify or engineer suitable Cas9homologs. According to one aspect, CRISPR-targetable sequences includethose having different PAM requirements (see reference (9)), or directedevolution. According to one aspect, inactivating one of the Cas9nuclease domains increases the ratio of HR to NHEJ and may reducetoxicity (FIGS. 3A-1 and 3A-2, FIG. 5) (4, 5), while inactivating bothdomains may enable Cas9 to function as a retargetable DNA bindingprotein. Embodiments of the present disclosure have broad utility insynthetic biology (see references (21, 22)), the direct and multiplexedperturbation of gene networks (see references (13, 23)), and targeted exvivo (see references (24-26)) and in vivo gene therapy (see reference(27)).

According to certain aspects, a “re-engineerable organism” is providedas a model system for biological discovery and in vivo screening.According to one aspect, a “re-engineerable mouse” bearing an inducibleCas9 transgene is provided, and localized delivery (usingadeno-associated viruses, for example) of libraries of gRNAs targetingmultiple genes or regulatory elements allow one to screen for mutationsthat result in the onset of tumors in the target tissue type. Use ofCas9 homologs or nuclease-null variants bearing effector domains (suchas activators) allow one to multiplex activate or repress genes in vivo.According to this aspect, one could screen for factors that enablephenotypes such as: tissue-regeneration, trans-differentiation etc.According to certain aspects, (a) use of DNA-arrays enables multiplexsynthesis of defined gRNA libraries (refer FIGS. 13A and 13B); and (b)gRNAs being small in size (refer FIG. 3b ) are packaged and deliveredusing a multitude of non-viral or viral delivery methods.

According to one aspect, the lower toxicities observed with “nickases”for genome engineering applications is achieved by inactivating one ofthe Cas9 nuclease domains, either the nicking of the DNA strandbase-paired with the RNA or nicking its complement. Inactivating bothdomains allows Cas9 to function as a retargetable DNA binding protein.According to one aspect, the Cas9 retargetable DNA binding protein isattached

(a) to transcriptional activation or repression domains for modulatingtarget gene expression, including but not limited to chromatinremodeling, histone modification, silencing, insulation, directinteractions with the transcriptional machinery;

(b) to nuclease domains such as Fold to enable ‘highly specific’ genomeediting contingent upon dimerization of adjacent gRNA-Cas9 complexes;

(c) to fluorescent proteins for visualizing genomic loci and chromosomedynamics; or

(d) to other fluorescent molecules such as protein or nucleic acid boundorganic fluorophores, quantum dots, molecular beacons and echo probes ormolecular beacon replacements;

(e) to multivalent ligand-binding protein domains that enableprogrammable manipulation of genome-wide 3D architecture.

According to one aspect, the transcriptional activation and repressioncomponents can employ CRISPR systems naturally or syntheticallyorthogonal, such that the gRNAs only bind to the activator or repressorclass of Cas. This allows a large set of gRNAs to tune multiple targets.

According to certain aspects, the use of gRNAs provide the ability tomultiplex than mRNAs in part due to the smaller size—100 vs. 2000nucleotide lengths respectively. This is particularly valuable whennucleic acid delivery is size limited, as in viral packaging. Thisenables multiple instances of cleavage, nicking, activation, orrepression—or combinations thereof. The ability to easily targetmultiple regulatory targets allows the coarse-or-fine-tuning orregulatory networks without being constrained to the natural regulatorycircuits downstream of specific regulatory factors (e.g. the 4 mRNAsused in reprogramming fibroblasts into IPSCs). Examples of multiplexingapplications include:

1. Establishing (major and minor) histocompatibility alleles,haplotypes, and genotypes for human (or animal) tissue/organtransplantation. This aspect results e.g. in HLA homozygous cell linesor humanized animal breeds—or —a set of gRNAs capable of superimposingsuch HLA alleles onto an otherwise desirable cell lines or breeds.2. Multiplex cis-regulatory element (CRE=signals for transcription,splicing, translation, RNA and protein folding, degradation, etc.)mutations in a single cell (or a collection of cells) can be used forefficiently studying the complex sets of regulatory interaction that canoccur in normal development or pathological, synthetic or pharmaceuticalscenarios. According to one aspect, the CREs are (or can be made)somewhat orthogonal (i.e. low cross talk) so that many can be tested inone setting—e.g. in an expensive animal embryo time series. Oneexemplary application is with RNA fluorescent in situ sequencing(FISSeq).3. Multiplex combinations of CRE mutations and/or epigenetic activationor repression of CREs can be used to alter or reprogram iPSCs or ESCs orother stem cells or non-stem cells to any cell type or combination ofcell types for use in organs-on-chips or other cell and organ culturesfor purposes of testing pharmaceuticals (small molecules, proteins,RNAs, cells, animal, plant or microbial cells, aerosols and otherdelivery methods), transplantation strategies, personalizationstrategies, etc.4. Making multiplex mutant human cells for use in diagnostic testing(and/or DNA sequencing) for medical genetics. To the extent that thechromosomal location and context of a human genome allele (or epigeneticmark) can influence the accuracy of a clinical genetic diagnosis, it isimportant to have alleles present in the correct location in a referencegenome—rather than in an ectopic (aka transgenic) location or in aseparate piece of synthetic DNA. One embodiment is a series ofindependent cell lines one per each diagnostic human SNP, or structuralvariant. Alternatively, one embodiment includes multiplex sets ofalleles in the same cell. In some cases multiplex changes in one gene(or multiple genes) will be desirable under the assumption ofindependent testing. In other cases, particular haplotype combinationsof alleles allows testing of sequencing (genotyping) methods whichaccurately establish haplotype phase (i.e. whether one or both copies ofa gene are affected in an individual person or somatic cell type.5. Repetitive elements or endogenous viral elements can be targeted withengineered Cas+gRNA systems in microbes, plants, animals, or human cellsto reduce deleterious transposition or to aid in sequencing or otheranalytic genomic/transcriptomic/proteomic/diagnostic tools (in whichnearly identical copies can be problematic).

The following references identified by number in the foregoing sectionare hereby incorporated by reference in their entireties.

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The following examples are set forth as being representative of thepresent disclosure. These examples are not to be construed as limitingthe scope of the present disclosure as these and other equivalentembodiments will be apparent in view of the present disclosure, figuresand accompanying claims.

Example I The Type II CRISPR-Cas System

According to one aspect, embodiments of the present disclosure utilizeshort RNA to identify foreign nucleic acids for activity by a nucleasein a eukaryotic cell. According to a certain aspect of the presentdisclosure, a eukaryotic cell is altered to include within its genomenucleic acids encoding one or more short RNA and one or more nucleaseswhich are activated by the binding of a short RNA to a target DNAsequence. According to certain aspects, exemplary short RNA/enzymesystems may be identified within bacteria or archaea, such as(CRISPR)/CRISPR-associated (Cas) systems that use short RNA to directdegradation of foreign nucleic acids. CRISPR (“clustered regularlyinterspaced short palindromic repeats”) defense involves acquisition andintegration of new targeting “spacers” from invading virus or plasmidDNA into the CRISPR locus, expression and processing of short guidingCRISPR RNAs (crRNAs) consisting of spacer-repeat units, and cleavage ofnucleic acids (most commonly DNA) complementary to the spacer.

Three classes of CRISPR systems are generally known and are referred toas Type I, Type II or Type III). According to one aspect, a particularuseful enzyme according to the present disclosure to cleave dsDNA is thesingle effector enzyme, Cas9, common to Type II. (See reference (1)).Within bacteria, the Type II effector system consists of a longpre-crRNA transcribed from the spacer-containing CRISPR locus, themultifunctional Cas9 protein, and a tracrRNA important for gRNAprocessing. The tracrRNAs hybridize to the repeat regions separating thespacers of the pre-crRNA, initiating dsRNA cleavage by endogenous RNaseIII, which is followed by a second cleavage event within each spacer byCas9, producing mature crRNAs that remain associated with the tracrRNAand Cas9. According to one aspect, eukaryotic cells of the presentdisclosure are engineered to avoid use of RNase III and the crRNAprocessing in general. See reference (2).

According to one aspect, the enzyme of the present disclosure, such asCas9 unwinds the DNA duplex and searches for sequences matching thecrRNA to cleave. Target recognition occurs upon detection ofcomplementarity between a “protospacer” sequence in the target DNA andthe remaining spacer sequence in the crRNA. Importantly, Cas9 cuts theDNA only if a correct protospacer-adjacent motif (PAM) is also presentat the 3′ end. According to certain aspects, differentprotospacer-adjacent motif can be utilized. For example, the S. pyogenessystem requires an NGG sequence, where N can be any nucleotide. S.thermophilus Type II systems require NGGNG (see reference (3)) andNNAGAAW (see reference (4)), respectively, while different S. mutanssystems tolerate NGG or NAAR (see reference (5)). Bioinformatic analyseshave generated extensive databases of CRISPR loci in a variety ofbacteria that may serve to identify additional useful PAMs and expandthe set of CRISPR-targetable sequences (see references (6, 7)). In S.thermophilus, Cas9 generates a blunt-ended double-stranded break 3 bpprior to the 3′ end of the protospacer (see reference (8)), a processmediated by two catalytic domains in the Cas9 protein: an HNH domainthat cleaves the complementary strand of the DNA and a RuvC-like domainthat cleaves the non-complementary strand (See FIG. 1A and FIGS. 3A-1and 3A-2). While the S. pyogenes system has not been characterized tothe same level of precision, DSB formation also occurs towards the 3′end of the protospacer. If one of the two nuclease domains isinactivated, Cas9 will function as a nickase in vitro (see reference(2)) and in human cells (see FIG. 5).

According to one aspect, the specificity of gRNA-directed Cas9 cleavageis used as a mechanism for genome engineering in a eukaryotic cell.According to one aspect, hybridization of the gRNA need not be 100percent in order for the enzyme to recognize the gRNA/DNA hybrid andaffect cleavage. Some off-target activity could occur. For example, theS. pyogenes system tolerates mismatches in the first 6 bases out of the20 bp mature spacer sequence in vitro. According to one aspect, greaterstringency may be beneficial in vivo when potential off-target sitesmatching (last 14 bp) NGG exist within the human reference genome forthe gRNAs. The effect of mismatches and enzyme activity in general aredescribed in references (9), (2), (10), and (4).

According to certain aspects, specificity may be improved. Wheninterference is sensitive to the melting temperature of the gRNA-DNAhybrid, AT-rich target sequences may have fewer off-target sites.Carefully choosing target sites to avoid pseudo-sites with at least 14bp matching sequences elsewhere in the genome may improve specificity.The use of a Cas9 variant requiring a longer PAM sequence may reduce thefrequency of off-target sites. Directed evolution may improve Cas9specificity to a level sufficient to completely preclude off-targetactivity, ideally requiring a perfect 20 bp gRNA match with a minimalPAM. Accordingly, modification to the Cas9 protein is a representativeembodiment of the present disclosure. As such, novel methods permittingmany rounds of evolution in a short timeframe (see reference (11) andenvisioned. CRISPR systems useful in the present disclosure aredescribed in references (12, 13).

Example II Plasmid Construction

The Cas9 gene sequence was human codon optimized and assembled byhierarchical fusion PCR assembly of 9 500 bp gBlocks ordered from IDT.FIGS. 3A-1 and 3A-2 for the engineered type II CRISPR system for humancells shows the expression format and full sequence of the cas9 geneinsert. The RuvC-like and HNH motifs, and the C-terminus SV40 NLS arerespectively highlighted by blue, brown and orange colors. Cas9_D10A wassimilarly constructed. The resulting full-length products were clonedinto the pcDNA3.3-TOPO vector (Invitrogen). The target gRNA expressionconstructs were directly ordered as individual 455 bp gBlocks from IDTand either cloned into the pCR-BluntII-TOPO vector (Invitrogen) or peramplified. FIG. 3B shows the U6 promoter based expression scheme for theguide RNAs and predicted RNA transcript secondary structure. The use ofthe U6 promoter constrains the 1^(st) position in the RNA transcript tobe a ‘G’ and thus all genomic sites of the form GN₂₀GG can be targetedusing this approach. FIG. 3C shows the 7 gRNAs used.

The vectors for the HR reporter assay involving a broken GFP wereconstructed by fusion PCR assembly of the GFP sequence bearing the stopcodon and 68 bp AAVS1 fragment (or mutants thereof; see FIGS. 6A and6B), or 58 bp fragments from the DNMT3a and DNMT3b genomic loci (seeFIG. 8A) assembled into the EGIP lentivector from Addgene (plasmid#26777). These lentivectors were then used to establish the GFP reporterstable lines. TALENs used in this study were constructed using theprotocols described in (14). All DNA reagents developed in this studyare available at Addgene.

Example III Cell Culture

PGP1 iPS cells were maintained on Matrigel (BD Biosciences)-coatedplates in mTeSR1 (Stemcell Technologies). Cultures were passaged every5-7 d with TrypLE Express (Invitrogen). K562 cells were grown andmaintained in RPMI (Invitrogen) containing 15% FBS. HEK 293T cells werecultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) highglucose supplemented with 10% fetal bovine serum (FBS, Invitrogen),penicillin/streptomycin (pen/strep, Invitrogen), and non-essential aminoacids (NEAA, Invitrogen). All cells were maintained at 37° C. and 5% CO₂in a humidified incubator.

Example IV Gene Targeting of PGP1 iPS, K562 and 293 Ts

PGP1 iPS cells were cultured in Rho kinase (ROCK) inhibitor (Calbiochem)2h before nucleofection. Cells were harvest using TrypLE Express(Invitrogen) and 2×10⁶ cells were resuspended in P3 reagent (Lonza) with1 μg Cas9 plasmid, 1 μg gRNA and/or 1 μg DNA donor plasmid, andnucleofected according to manufacturer's instruction (Lonza). Cells weresubsequently plated on an mTeSR1-coated plate in mTeSR1 mediumsupplemented with ROCK inhibitor for the first 24 h. For K562s, 2×10⁶cells were resuspended in SF reagent (Lonza) with 1 μg Cas9 plasmid, 1μg gRNA and/or 1 μg DNA donor plasmid, and nucleofected according tomanufacturer's instruction (Lonza). For 293 Ts, 0.1×10⁶ cells weretransfected with 1 μg Cas9 plasmid, 1 μg gRNA and/or 1 μg DNA donorplasmid using Lipofectamine 2000 as per the manufacturer's protocols.The DNA donors used for endogenous AAVS1 targeting were either a dsDNAdonor (FIG. 2C) or a 90mer oligonucleotide. The former has flankingshort homology arms and a SA-2A-puromycin-CaGGS-eGFP cassette to enrichfor successfully targeted cells.

The targeting efficiency was assessed as follows. Cells were harvested 3days after nucleofection and the genomic DNA of ˜1×10⁶ cells wasextracted using prepGEM (ZyGEM). PCR was conducted to amplify thetargeting region with genomic DNA derived from the cells and ampliconswere deep sequenced by MiSeq Personal Sequencer (Illumina) withcoverage >200,000 reads. The sequencing data was analyzed to estimateNHEJ efficiencies. The reference AAVS1 sequence analyzed is:

(SEQ ID NO: 1) CACTTCAGGACAGCATGTTTGCTGCCTCCAGGGATCCTGTGTCCCCGAGCTGGGACCACCTTATATTCCCAGGGCCGGTTAATGTGGCTCTGGTTCTGGGTACTTTTATCTGTCCCCTCCACCCCACAGTGGGGCCACTAGGGACAGGATTGGTGACAGAAAAGCCCCATCCTTAGGCCTCCTCCTTCCTAGTCTCCTGATATTGGGTCTAACCCCCACCTCCTGTTAGGCAGATTCCTTATCTGGTGACACACCCCCATTTCCTGGAThe PCR primers for amplifying the targeting regions in the human genomeare:

AAVS1-R (SEQ ID NO: 2)CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTacaggaggtgggggttagac AAVS1-F.1(SEQ ID NO: 3)ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATtatattcccagggccggtta AAVS1-F.2(SEQ ID NO: 4)ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGtatattcccagggccggtta AAVS1-F.3(SEQ ID NO: 5)ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCTAAtatattcccagggccggtta AAVS1-F.4(SEQ ID NO: 6)ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGTCAtatattcccagggccggtta AAVS1-F.5(SEQ ID NO: 7)ACACTCTTTCCCTACACGACGCTCTTCCGATCTCACTGTtatattcccagggccggtta AAVS1-F.6(SEQ ID NO: 8)ACACTCTTTCCCTACACGACGCTCTTCCGATCTATTGGCtatattcccagggccggtta AAVS1-F.7(SEQ ID NO: 9)ACACTCTTTCCCTACACGACGCTCTTCCGATCTGATCTGtatattcccagggccggtta AAVS1-F.8(SEQ ID NO: 10)ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCAAGTtatattcccagggccggtta AAVS1-F.9(SEQ ID NO: 11)ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGATCtatattcccagggccggtta AAVS1-F.10(SEQ ID NO: 12)ACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGCTAtatattcccagggccggtta AAVS1-F.11(SEQ ID NO: 13)ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTAGCCtatattcccagggccggtta AAVS1-F.12(SEQ ID NO: 14)ACACTCTTTCCCTACACGACGCTCTTCCGATCTTACAAGtatattcccagggccggtta

To analyze the HR events using the DNA donor in FIG. 2C, the primersused were:

(SEQ ID NO: 15) HR_AAVS1-F CTGCCGTCTCTCTCCTGAGT (SEQ ID NO: 16)HR_Puro-R GTGGGCTTGTACTCGGTCAT

Example V Bioinformatics Approach for Computing Human Exon CRISPRTargets and Methodology for their Multiplexed Synthesis

A set of gRNA gene sequences that maximally target specific locations inhuman exons but minimally target other locations in the genome weredetermined as follows. According to one aspect, maximally efficienttargeting by a gRNA is achieved by 23 nt sequences, the 5′-most 20 nt ofwhich exactly complement a desired location, while the three 3′-mostbases must be of the form NGG. Additionally, the 5′-most nt must be a Gto establish a pol-III transcription start site. However, according to(2), mispairing of the six 5′-most nt of a 20 bp gRNA against itsgenomic target does not abrogate Cas9-mediated cleavage so long as thelast 14 nt pairs properly, but mispairing of the eight 5′-most nt alongwith pairing of the last 12 nt does, while the case of the seven 5-mostnt mispairs and 13 3′ pairs was not tested. To be conservative regardingoff-target effects, one condition was that the case of the seven 5′-mostmispairs is, like the case of six, permissive of cleavage, so thatpairing of the 3′-most 13 nt is sufficient for cleavage. To identifyCRISPR target sites within human exons that should be cleavable withoutoff-target cuts, all 23 bp sequences of the form 5′-GBBBB BBBBB BBBBBBBBBB NGG-3′ (form 1) were examined, where the B's represent the basesat the exon location, for which no sequence of the form 5′-NNNNN NNBBBBBBBB BBBBB NGG-3′ (form 2) existed at any other location in the humangenome. Specifically, (i) a BED file of locations of coding regions ofall RefSeq genes the GRCh37/hg19 human genome from the UCSC GenomeBrowser (15-17) was downloaded. Coding exon locations in this BED filecomprised a set of 346089 mappings of RefSeq mRNA accessions to the hg19genome. However, some RefSeq mRNA accessions mapped to multiple genomiclocations (probable gene duplications), and many accessions mapped tosubsets of the same set of exon locations (multiple isoforms of the samegenes). To distinguish apparently duplicated gene instances andconsolidate multiple references to the same genomic exon instance bymultiple RefSeq isoform accessions, (ii) unique numerical suffixes to705 RefSeq accession numbers that had multiple genomic locations wereadded, and (iii) the mergeBed function of BEDTools (18)(v2.16.2-zip-87e3926) was used to consolidate overlapping exon locationsinto merged exon regions. These steps reduced the initial set of 346089RefSeq exon locations to 192783 distinct genomic regions. The hg19sequence for all merged exon regions were downloaded using the UCSCTable Browser, adding 20 bp of padding on each end. (iv) Using customperl code, 1657793 instances of form 1 were identified within thisexonic sequence. (v) These sequences were then filtered for theexistence of off-target occurrences of form 2: For each merged exon form1 target, the 3′-most 13 bp specific (B) “core” sequences were extractedand, for each core generated the four 16 bp sequences 5′-BBB BBBBB BBBBBNGG-3′ (N=A, C, G, and T), and searched the entire hg19 genome for exactmatches to these 6631172 sequences using Bowtie version 0.12.8 (19)using the parameters −116-v 0-k 2. Any exon target site for which therewas more than a single match was rejected. Note that because anyspecific 13 bp core sequence followed by the sequence NGG confers only15 bp of specificity, there should be on average ˜5.6 matches to anextended core sequence in a random ˜3 Gb sequence (both strands).Therefore, most of the 1657793 initially identified targets wererejected; however 189864 sequences passed this filter. These comprisethe set of CRISPR-targetable exonic locations in the human genome. The189864 sequences target locations in 78028 merged exonic regions (40.5%of the total of 192783 merged human exon regions) at a multiplicity of˜2.4 sites per targeted exonic region. To assess targeting at a genelevel, RefSeq mRNA mappings were clustered so that any two RefSeqaccessions (including the gene duplicates distinguished in (ii)) thatoverlap a merged exon region are counted as a single gene cluster, the189864 exonic specific CRISPR sites target 17104 out of 18872 geneclusters (90.6% of all gene clusters) at a multiplicity of ˜11.1 pertargeted gene cluster. (Note that while these gene clusters collapseRefSeq mRNA accessions that represent multiple isoforms of a singletranscribed gene into a single entity, they will also collapseoverlapping distinct genes as well as genes with antisense transcripts.)At the level of original RefSeq accessions, the 189864 sequencestargeted exonic regions in 30563 out of a total of 43726 (69.9%) mappedRefSeq accessions (including distinguished gene duplicates) at amultiplicity of ˜6.2 sites per targeted mapped RefSeq accession.

According to one aspect, the database can be refined by correlatingperformance with factors, such as base composition and secondarystructure of both gRNAs and genomic targets (20, 21), and the epigeneticstate of these targets in human cell lines for which this information isavailable (22).

Example VI Multiplex Synthesis

The target sequences were incorporated into a 200 bp format that iscompatible for multiplex synthesis on DNA arrays (23, 24). According toone aspect the method allows for targeted retrieval of a specific orpools of gRNA sequences from the DNA array based oligonucleotide pooland its rapid cloning into a common expression vector (FIGS. 13A-1 and13A-2). Specifically, a 12 k oligonucleotide pool from CustomArray Inc.was synthesized. Furthermore, gRNAs of choice from this library (FIG.13B) were successfully retrieved. We observed an error rate of ˜4mutations per 1000 bp of synthesized DNA.

Example VII RNA-Guided Genome Editing Requires Both Cas9 and Guide RNAfor Successful Targeting

Using the GFP reporter assay described in FIG. 1B, all possiblecombinations of the repair DNA donor, Cas9 protein, and gRNA were testedfor their ability to effect successful HR (in 293 Ts). As shown in FIG.4, GFP+ cells were observed only when all the 3 components were present,validating that these CRISPR components are essential for RNA-guidedgenome editing. Data is mean+/−SEM (N=3).

Example VIII Analysis of gRNA and Cas9 Mediated Genome Editing

The CRISPR mediated genome editing process was examined using either (A)a GFP reporter assay as described earlier results of which are shown inFIG. 5A, and (B) deep sequencing of the targeted loci (in 293 Ts),results of which are shown in FIG. 5B. As comparison, a D10A mutant forCas9 was tested that has been shown in earlier reports to function as anickase in in vitro assays. As shown in FIG. 5, both Cas9 and Cas9D10Acan effect successful HR at nearly similar rates. Deep sequencinghowever confirms that while Cas9 shows robust NHEJ at the targeted loci,the D10A mutant has significantly diminished NHEJ rates (as would beexpected from its putative ability to only nick DNA). Also, consistentwith the known biochemistry of the Cas9 protein, NHEJ data confirms thatmost base-pair deletions or insertions occurred near the 3′ end of thetarget sequence: the peak is ˜3-4 bases upstream of the PAM site, with amedian deletion frequency of ˜9-10 bp. Data is mean+/−SEM (N=3).

Example IX RNA-Guided Genome Editing is Target Sequence Specific

Similar to the GFP reporter assay described in FIG. 1B, 3 293T stablelines each bearing a distinct GFP reporter construct were developed.These are distinguished by the sequence of the AAVS1 fragment insert (asindicated in the FIGS. 6A and 6B). One line harbored the wild-typefragment while the two other lines were mutated at 6 bp (highlighted inred). Each of the lines was then targeted by one of the following 4reagents: a GFP-ZFN pair that can target all cell types since itstargeted sequence was in the flanking GFP fragments and hence present inalong cell lines; a AAVS1 TALEN that could potentially target only thewt-AAVS1 fragment since the mutations in the other two lines shouldrender the left TALEN unable to bind their sites; the T1 gRNA which canalso potentially target only the wt-AAVS1 fragment, since its targetsite is also disrupted in the two mutant lines; and finally the T2 gRNAwhich should be able to target all 3 cell lines since, unlike the T1gRNA, its target site is unaltered among the 3 lines. ZFN modified all 3cell types, the AAVS1 TALENs and the T1 gRNA only targeted the wt-AAVS1cell type, and the T2 gRNA successfully targets all 3 cell types. Theseresults together confirm that the guide RNA mediated editing is targetsequence specific. Data is mean+/−SEM (N=3).

Example X Guide RNAs Targeted to the GFP Sequence Enable Robust GenomeEditing

In addition to the 2 gRNAs targeting the AAVS1 insert, two additionalgRNAs targeting the flanking GFP sequences of the reporter described inFIG. 1B (in 293 Ts) were tested. As shown in FIG. 7, these gRNAs werealso able to effect robust HR at this engineered locus. Data ismean+/−SEM (N=3).

Example XI RNA-Guided Genome Editing is Target Sequence Specific, andDemonstrates Similar Targeting Efficiencies as ZFNs or TALENs

Similar to the GFP reporter assay described in FIG. 1B, two 293T stablelines each bearing a distinct GFP reporter construct were developed.These are distinguished by the sequence of the fragment insert (asindicated in FIG. 8A). One line harbored a 58 bp fragment from theDNMT3a gene while the other line bore a homologous 58 bp fragment fromthe DNMT3b gene. The sequence differences are highlighted in red. Eachof the lines was then targeted by one of the following 6 reagents: aGFP-ZFN pair that can target all cell types since its targeted sequencewas in the flanking GFP fragments and hence present in along cell lines;a pair of TALENs that potentially target either DNMT3a or DNMT3bfragments; a pair of gRNAs that can potentially target only the DNMT3afragment; and finally a gRNA that should potentially only target theDNMT3b fragment. As indicated in FIG. 8B, the ZFN modified all 3 celltypes, and the TALENs and gRNAs only their respective targets.Furthermore the efficiencies of targeting were comparable across the 6targeting reagents. These results together confirm that RNA-guidedediting is target sequence specific and demonstrates similar targetingefficiencies as ZFNs or TALENs. Data is mean+/−SEM (N=3).

Example XII RNA-Guided NHEJ in Human iPS Cells

Human iPS cells (PGP1) were nucleofected with constructs indicated inthe left panel of FIGS. 9A, 9B, and 9C. 4 days after nucleofection, NHEJrate was measured by assessing genomic deletion and insertion rate atdouble-strand breaks (DSBs) by deep sequencing. Panel 1: Deletion ratedetected at targeting region. Red dash lines: boundary of T1 RNAtargeting site; green dash lines: boundary of T2 RNA targeting site. Thedeletion incidence at each nucleotide position was plotted in blacklines and the deletion rate as the percentage of reads carryingdeletions was calculated. Panel 2: Insertion rate detected at targetingregion. Red dash lines: boundary of T1 RNA targeting site; green dashlines: boundary of T2 RNA targeting site. The incidence of insertion atthe genomic location where the first insertion junction was detected wasplotted in black lines and the insertion rate as the percentage of readscarrying insertions was calculated. Panel 3: Deletion size distribution.The frequencies of different size deletions among the whole NHEJpopulation was plotted. Panel 4: insertion size distribution. Thefrequencies of different sizes insertions among the whole NHEJpopulation was plotted. iPS targeting by both gRNAs is efficient (2-4%),sequence specific (as shown by the shift in position of the NHEJdeletion distributions), and reaffirming the results of FIG. 4, theNGS-based analysis also shows that both the Cas9 protein and the gRNAare essential for NHEJ events at the target locus.

Example XIII RNA-Guided NHEJ in K562 Cells

K562 cells were nucleated with constructs indicated in the left panel ofFIGS. 10A and 10B. 4 days after nucleofection, NHEJ rate was measured byassessing genomic deletion and insertion rate at DSBs by deepsequencing. Panel 1: Deletion rate detected at targeting region. Reddash lines: boundary of T1 RNA targeting site; green dash lines:boundary of T2 RNA targeting site. The deletion incidence at eachnucleotide position was plotted in black lines and the deletion rate asthe percentage of reads carrying deletions was calculated. Panel 2:Insertion rate detected at targeting region. Red dash lines: boundary ofT1 RNA targeting site; green dash lines: boundary of T2 RNA targetingsite. The incidence of insertion at the genomic location where the firstinsertion junction was detected was plotted in black lines and theinsertion rate as the percentage of reads carrying insertions wascalculated. Panel 3: Deletion size distribution. The frequencies ofdifferent size deletions among the whole NHEJ population was plotted.Panel 4: insertion size distribution. The frequencies of different sizesinsertions among the whole NHEJ population was plotted. K562 targetingby both gRNAs is efficient (13-38%) and sequence specific (as shown bythe shift in position of the NHEJ deletion distributions). Importantly,as evidenced by the peaks in the histogram of observed frequencies ofdeletion sizes, simultaneous introduction of both T1 and T2 guide RNAsresulted in high efficiency deletion of the intervening 19 bp fragment,demonstrating that multiplexed editing of genomic loci is also feasibleusing this approach.

Example XIV RNA-Guided NHEJ in 293T Cells

293T cells were transfected with constructs indicated in the left panelof FIGS. 11A and 11B. 4 days after nucleofection, NHEJ rate was measuredby assessing genomic deletion and insertion rate at DSBs by deepsequencing. Panel 1: Deletion rate detected at targeting region. Reddash lines: boundary of T1 RNA targeting site; green dash lines:boundary of T2 RNA targeting site. The deletion incidence at eachnucleotide position was plotted in black lines and the deletion rate asthe percentage of reads carrying deletions was calculated. Panel 2:Insertion rate detected at targeting region. Red dash lines: boundary ofT1 RNA targeting site; green dash lines: boundary of T2 RNA targetingsite. The incidence of insertion at the genomic location where the firstinsertion junction was detected was plotted in black lines and theinsertion rate as the percentage of reads carrying insertions wascalculated. Panel 3: Deletion size distribution. The frequencies ofdifferent size deletions among the whole NHEJ population was plotted.Panel 4: insertion size distribution. The frequencies of different sizesinsertions among the whole NHEJ population was plotted. 293T targetingby both gRNAs is efficient (10-24%) and sequence specific (as shown bythe shift in position of the NHEJ deletion distributions).

Example XV HR at the Endogenous AAVS1 Locus Using Either a dsDNA Donoror a Short Oligonucleotide Donor

As shown in FIG. 12A, PCR screen (with reference to FIG. 2C) confirmedthat 21/24 randomly picked 293T clones were successfully targeted. Asshown in FIG. 12B, similar PCR screen confirmed 3/7 randomly pickedPGP1-iPS clones were also successfully targeted. As shown in FIG. 12C,short 90mer oligos could also effect robust targeting at the endogenousAAVS1 locus (shown here for K562 cells).

Example XVI Methodology for Multiplex Synthesis, Retrieval and U6Expression Vector Cloning of Guide RNAs Targeting Genes in the HumanGenome

A resource of about 190 k bioinformatically computed unique gRNA sitestargeting ˜40.5% of all exons of genes in the human genome wasgenerated. As shown in FIG. 13A-1 and FIG. 13A-2, the gRNA target siteswere incorporated into a 200 bp format that is compatible for multiplexsynthesis on DNA arrays. Specifically, the design allows for (i)targeted retrieval of a specific or pools of gRNA targets from the DNAarray oligonucleotide pool (through 3 sequential rounds of nested PCR asindicated in the figure schematic); and (ii) rapid cloning into a commonexpression vector which upon linearization using an AflII site serves asa recipient for Gibson assembly mediated incorporation of the gRNAinsert fragment. As shown in FIG. 13B, the method was used to accomplishtargeted retrieval of 10 unique gRNAs from a 12 k oligonucleotide poolsynthesized by CustomArray Inc.

Example XVII CRISPR Mediated RNA-Guided Transcriptional Activation

The CRISPR-Cas system has an adaptive immune defense system in bacteriaand functions to ‘cleave’ invading nucleic acids. According to oneaspect, the CRISPR-CAS system is engineered to function in human cells,and to ‘cleave’ genomic DNA. This is achieved by a short guide RNAdirecting a Cas9 protein (which has nuclease function) to a targetsequence complementary to the spacer in the guide RNA. The ability to‘cleave’ DNA enables a host of applications related to genome editing,and also targeted genome regulation. Towards this, the Cas9 protein wasmutated to make it nuclease-null by introducing mutations that arepredicted to abrogate coupling to Mg2+(known to be important for thenuclease functions of the RuvC-like and HNH-like domains): specifically,combinations of D10A, D839A, H840A and N863A mutations were introduced.The thus generated Cas9 nuclease-null protein (as confirmed by itsability to not cut DNA by sequencing analysis) and hereafter referred toas Cas9R-H-, was then coupled to a transcriptional activation domain,here VP64, enabling the CRISPR-cas system to function as a RNA guidedtranscription factor (see FIGS. 14A-D). The Cas9R-H-+VP64 fusion enablesRNA-guided transcriptional activation at the two reporters shown.Specifically, both FACS analysis and immunofluorescence imagingdemonstrates that the protein enables gRNA sequence specific targetingof the corresponding reporters, and furthermore, the resultingtranscription activation as assayed by expression of a dTomatofluorescent protein was at levels similar to those induced by aconvention TALE-VP64 fusion protein.

Example XVIII gRNA Sequence Flexibility and Applications Thereof

Flexibility of the gRNA scaffold sequence to designer sequenceinsertions was determined by systematically assaying for a range of therandom sequence insertions on the 5′, middle and 3′ portions of thegRNA: specifically, 1 bp, 5 bp, 10 bp, 20 bp, and 40 bp inserts weremade in the gRNA sequence at the 5′, middle, and 3′ ends of the gRNA(the exact positions of the insertion are highlighted in ‘red’ in FIG.15A). This gRNA was then tested for functionality by its ability toinduce HR in a GFP reporter assay (as described herein). It is evidentthat gRNAs are flexible to sequence insertions on the 5′ and 3′ ends (asmeasured by retained HR inducing activity). Accordingly, aspects of thepresent disclosure are directed to tagging of small-molecule responsiveRNA aptamers that may trigger onset of gRNA activity, or gRNAvisualization. Additionally, aspects of the present disclosure aredirected to tethering of ssDNA donors to gRNAs via hybridization, thusenabling coupling of genomic target cutting and immediate physicallocalization of repair template which can promote homologousrecombination rates over error-prone non-homologous end-joining.

The following references identified in the Examples section by numberare hereby incorporated by reference in their entireties for allpurposes.

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The invention claimed is:
 1. A method of altering a eukaryotic cellcomprising providing the eukaryotic cell with a guide RNA having aspacer sequence complementary to genomic DNA of the eukaryotic cell anda scaffold sequence, providing the eukaryotic cell with a codonoptimized Cas9 that interacts with the guide RNA and cleaves the genomicDNA in a site specific manner, wherein the guide RNA binds tocomplementary genomic DNA and the codon optimized Cas9 cleaves thegenomic DNA in a site specific manner, and wherein the codon optimizedCas9 is a human codon optimized Cas9 encoded by a nucleic acidcomprising SEQ ID NO:22.
 2. The method of claim 1 wherein the eukaryoticcell is provided with the guide RNA by introducing into the eukaryoticcell a nucleic acid encoding the guide RNA and wherein the eukaryoticcell is provided with the codon optimized Cas9 by introducing into theeukaryotic cell a nucleic acid encoding the codon optimized Cas9.
 3. Themethod of claim 1 wherein the eukaryotic cell is a yeast cell, a plantcell or a mammalian cell.
 4. The method of claim 1 wherein the guide RNAincludes between about 10 to about 250 nucleotides.
 5. The method ofclaim 1 wherein the guide RNA includes between about 20 to about 100nucleotides.
 6. The method of claim 1 wherein the eukaryotic cell is ahuman cell.
 7. A method of altering a eukaryotic cell at a plurality ofgenomic DNA sites comprising providing the eukaryotic cell with aplurality of guide RNAs, each guide RNA having a spacer sequencecomplementary to a different site on genomic DNA of the eukaryotic celland a scaffold sequence, providing the eukaryotic cell with a codonoptimized Cas9, wherein the guide RNAs bind to complementary genomic DNAand the codon optimized Cas9 cleaves the genomic DNA in a site specificmanner, and wherein the codon optimized Cas9 is a human codon optimizedCas9 encoded by a nucleic acid comprising SEQ ID NO:22.
 8. The method ofclaim 7 wherein the eukaryotic cell is provided with the plurality ofguide RNAs by introducing into the eukaryotic cell a plurality ofnucleic acids encoding the guide RNAs and wherein the eukaryotic cell isprovided with the codon optimized Cas9 by introducing into theeukaryotic cell a nucleic acid encoding the codon optimized Cas9.
 9. Themethod of claim 7 wherein the eukaryotic cell is a yeast cell, a plantcell or a mammalian cell.
 10. The method of claim 7 wherein the guideRNA includes between about 10 to about 250 nucleotides.
 11. The methodof claim 7 wherein the guide RNA includes between about 20 to about 100nucleotides.
 12. The method of claim 7 wherein the eukaryotic cell is ahuman cell.